本文選題：紫花苜蓿 + 擬南芥��； 參考：《中國農業(yè)科學院》2013年碩士論文

【摘要】：草甘膦（Glyphosate）商品名稱為農達，是一種廣譜高效的滅生性除草劑，在農業(yè)生產(chǎn)中被廣泛應用。紫花苜蓿（Medicago sativa L.）是一種多年生豆科飼草作物，有“牧草之王”的美譽，在我國種植廣泛。農田雜草的滋生，不僅增加了成本投入，而且影響了牧草的產(chǎn)量和品質。本研究將具有自主知識產(chǎn)權的抗草甘膦基因GR79Mt轉入擬南芥和苜蓿中，以期獲得具有草甘膦抗性的植株。 本實驗得出如下結果： 1.建立了中苜6號組織培養(yǎng)再生體系。以下胚軸為外植體愈傷組織誘導率要高于以子葉為外植體的誘導率。在含2mg/L2,4-D和0.2mg/L KT的SH培養(yǎng)基上誘導愈傷組織效率最高，而分化培養(yǎng)基中僅含0.2mg/L的KT即可高效地誘導出胚狀體。同時，1g/L水解酪蛋白的加入，對胚狀體的產(chǎn)生和成熟有重要作用。胚狀體或小植株在不含植物激素的1/2MS培養(yǎng)基中誘導生根。 2.根據(jù)已知的抗草甘膦GR79Mt基因序列，在其前端加入葉綠體轉運肽基因(CTP2)序列，由增強型啟動子CAMV35S啟動轉錄，豌豆rbcS E9基因的多聚腺嘌呤序列終止轉錄。遺傳元件構建完成后人工合成序列，兩端加入PvuⅠ酶切位點。植物表達載體pCAMBIA2300用PvuⅠ酶切后留下原核表達框，與合成序列連接，成功的構建了表達載體pCA-GM。 3.通過農桿菌介導法轉化擬南芥花序，將收獲的種子在含60mg/L草甘膦的MS培養(yǎng)基篩選，獲得T1代植株。提取其基因組DNA進行PCR、RT-PCR驗證，目的基因可在轉錄水平上表達，說明目的基因已整合到擬南芥基因組上。 4.通過農桿菌介導的雙邊界雙元載體pCA-GM，，轉化中苜6號愈傷組織。誘導培養(yǎng)基中含2mg/L2,4-D、0.2mg/L KT和60mg/L的草甘膦。誘導胚狀體時，KT濃度調整為0.4mg/L，草甘膦濃度增加至80mg/L。在無植物生長調節(jié)劑的1/2MS培養(yǎng)基中誘導生根。最后獲得12株苜蓿再生植株，經(jīng)PCR驗證兩株呈陽性，初步表明目的基因已整合到苜蓿基因組中。
[Abstract]:Glyphosate (Glyphosate) is a broad-spectrum and high-efficient herbicide, which is widely used in agricultural production. Medicago sativa L. Is a perennial legume forage crop, with the "King of forage" reputation, widely planted in China. The breeding of weeds in farmland not only increases the cost input, but also affects the yield and quality of forage grass. In this study, glyphosate resistant gene GR79Mt with independent intellectual property rights was transferred into Arabidopsis thaliana and alfalfa in order to obtain glyphosate resistant plants. The results of this experiment are as follows: 1. The regeneration system of tissue culture of Zhongmu No.6 was established. The callus induction rate of cotyledons was higher than that of cotyledon explants. The callus induction efficiency was the highest on SH medium containing 2mg / L _ 2N _ 4-D and 0.2mg/L KT, but the embryoid was induced efficiently by KT containing only 0.2mg/L in the differentiation medium. At the same time, the addition of 1 g / L hydrolyzed casein plays an important role in the formation and maturation of embryoid. Embryoids or small plants were induced to take root in 1/2MS medium without plant hormones. 2. According to the known glyphosate-resistant GR79Mt gene sequence, the chloroplast transporter peptide gene (CTP2) sequence was added to the front of the gene, and the transcription was initiated by the enhanced promoter CAMV35S, and the polyadenine sequence of the pea rbcS E9 gene was terminated. After the construction of the genetic element, the synthetic sequence was constructed and the Pvu 鈪

本文編號：1938780